Field of the Invention
The present invention relates to polypeptides having cellulolytic activity or hemicellulolytic activity and polynucleotides encoding the polypeptides. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
Description of the Related Art
There is a rising demand for more sustainable solutions to important problems of a modern society. Demand for more biological processes, products and solutions: Post peak oil era makes it inevitable that the global society at large will have to change from being based on carbon resources from fossils to using renewables. Renewable carbon resources are primarily made of plant materials. The conversion of plant materials (renewable carbon) to substitute the spectrum of useful and needed products (as energy, plastics, chemicals, etc.) obtained currently from crude oil is in general achieved by conversion of the plant biopolymers by the help of microbial enzymes/proteins. This need places high demands on discovery of enzymes and auxiliary proteins from microbes, sufficiently diverse and efficient for converting q wide spectrum of different types of biomass, available globally: from corn stover and wheat straw over sugar cane bagasse and empty flower bunches of oil palm to municipality waste and agroindustrial side streams.
The complexity of the biomass available places high demands to the microbial products: Most agricultural products will have to be reserved for feeding 9 billion people as well as for feeding animals for the food chain. The biomass available for industrial purposes will largely in the future be crop residue/biowaste materials. Such materials are primarily composed of plant lignocelluloses, a highly recalcitrant structure which needs a host of enzymes for full decomposition. This requires even higher demands on the discovery of new and improved enzymes of microbial origin.
An efficient way of enhancing the conversion rate of cellulosic feedstock into ethanol is raising the temperature but this strategy is limited by the temperature stability of the available enzymes.
Another way of enhancing the conversion rate of cellulosic feedstock into ethanol is to optimize the pretreatment of the feedstock before enzymatic degradation but this strategy is limited to acidic pretreatment methods by the pH optimum of the available enzymes.
A third way of enhancing the conversion rate of cellulosic feedstocks into ethanol is adding polypeptides that enhance the cellulolytic activity at low temperatures.
It would be advantageous in the art to improve the conversion of cellulosic feedstock with polypeptides with cellulolytic activity at high temperatures and to provide polypeptides with cellulolytic activity that would be compatible with pretreatment at high pH values. Furthermore, it would be advantageous in the art to improve the conversion of cellulosic feedstocks with polypeptides with cellulolytic enhancing activity at high temperatures. However, only one polypeptide with cellulolytic enhancing activity at high temperatures is known (U.S. Pat. No. 7,271,244). We have identified a number of thermophilic fungi that produce extracellular cellulase activity (endoglucanase and cellobiohydrolase activity) with higher thermostability than cellulase activity from other thermophilic fungi. In addition, several of the fungi produce cellulases that are highly active at pH values over 7. As the fungi produce cellulases with high thermostability and interesting pH optima it is reasonable to assume that other secreted enzymes from these fungi will also have higher thermostability and pH optima than normally observed for fungal enzymes. Therefore, these fungi are useful sources of new enzymes for industrial or other applications,
Protein and enzyme discovery can be based on genome sequencing (confined to one organism at a time and depends on time consuming annotations), activity screening (requiring cloning and available high throughput assays), and searching for novelty through sequence similarity (e.g., a PCR based approach).
For decomposition of cellulose and hemicelluloses it is rather simple to construct suitable PCR primers for discovering novel xylanases (e.g., GH10 and GH11) and novel endoglucanases (e.g., GH45) by PCR based screening. The 3D protein structure has through evolution maintained longer stretches of rather highly conserved regions, suitable for primer construction. However, other needed types of enzymes for cellulose decomposition such as the cellobiohydrolases or the auxiliary proteins belonging to GH61 group have either very high sequence variation within each protein family and/or limited areas of sufficient conservation or sequence similarity.
The motivation for the present invention is for more efficient PCR based discovery. The basis is a belief that it should be possible to construct primers based on further similarities than what is possible from an alignment approach simply nested in the fact that it has been possible to group enzymes and other proteins in protein families, which embrace proteins of even very low sequence similarity but with important similarities in fold and characteristics/activities. Similarly, such is also based on the fact that an in silico Blast search could identify a series of proteins which are only distantly related sequence wise but sharing characteristics as, for example, grouping in the same protein family (Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696).
Our hypothesis was that such possible regions suitable for primer construction could be identified based on bringing forward an advanced level of pattern recognition. This approach resulted in a method with simplicity on the one hand and on the other hand significant valuable advantages, such as speed.
The present invention provides polypeptides having cellulolytic activity or hemicellulolytic activity and polynucleotides encoding the polypeptides.